Potentiation Of Insulin Release Research Articles

Secretin N-terminal hexapeptide potentiates insulin release in mouse islets

Peptides representing the N-terminal part of secretin were synthesized and their effects tested on column-perifused isolated mouse pancreatic islets. Insulin release induced by d-glucose was potentiated by the two peptides His-Ser-Asp-Gly-Thr-Phe-OMe (S 1–6) and Ser-Asp-Gly-Thr-Phe-OMe (S 2–6). The consecutive smaller N-terminal peptides Asp-Gly-Thr-Phe-OMe (S 3–6) and Gly-Thr-Phe-OMe (S 4–6) had no effects while the dipeptide ester Thr-Phe-OMe (S 5–6) also potentiated the release of insulin. The results suggest that the N-terminal part of secretin may be involved in the marked in vitro glucose-dependent insulin release induced by secretin.

Dibutyryl Guanosine 3′,5′-Monophosphate Inhibits Cholecystokinin Potentiation of Insulin Release in the Isolated Perfused Rat Pancreas

(Bu)2cGMP is known to act as a specific competitive inhibitor for gastrin and cholecystokinin (CCK) peptides. We have examined the effects of (Bu)2cGMP on CCK octapeptide (CCK-8) stimulation of insulin release in the isolated perfused pancreas and compared them with those on protein output. Addition of (Bu)2cGMP after a 20-min perfusion with 100 pM CCK-8 resulted in two distinctly different phases of insulin suppression. There was a sharp initial decline in insulin release for 3 min, followed by transient recovery toward the control level for 5 min, and then a small decline until termination of (Bu)2cGMP infusion. (Bu)2cGMP produced a concentration-dependent inhibition of both phases of insulin decrement. (Bu)2cGMP also produced a concentration-dependent inhibition of protein output. Addition of 1 mM (Bu)2cGMP rapidly and completely abolished CCK-8-stimulated protein output. Since CCK is released by meal intake and exogenous CCK stimulates insulin release and augments glucose-induced insulin release, it is possible that endogenous CCK plays an important role in the enteroinsular axis. The present findings of blockade of CCK-8-induced insulin release by selective antagonist of the action of CCK provide evidence for CCK as a mediator in the enteroinsular axis.

Improved glucose tolerance in rats treated with oxazolidinediones.

5-(2-Chloro-6-methoxyphenyl)oxazolidine-2,4-dione (49) is the most potent agent selected from a series of 5-substituted oxazolidinediones that were found to cause improvements in glucose tolerance in previously fasted rats and potentiation of insulin release in response to a glucose challenge. These compounds were unique in not producing hypoglycemia below the normal fasting glycemia levels. Substituent effects at positions 2-6 of the phenyl ring were investigated. Optimal positions for substitution were found to be the 2-, 5-, and 6-positions. Variations in the oxazolidinedione ring generally lead to loss of activity. The synthesis and structure-activity relationships of this series are detailed.

The non-sulfonylurea moiety of gliquidone mimics the effects of the parent molecule on pancreatic B-cells.

Compound UL-DF 9 corresponds to the non-sulfonylurea moiety of gliquidone, a hypoglycaemic sulfonylurea of the second generation. Its effects on the B-cell function were studied in vitro with mouse islets. In the presence of a non-stimulatory concentration of glucose (3 mM), UL-DF 9 decreased 86Rb+ efflux and accelerated 45Ca2+ efflux from islet cells, depolarized the B-cell membrane and induced an electrical activity similar to that triggered by stimulatory concentrations of glucose, and increased insulin release. The changes in 45Ca2+ efflux and insulin release, but not the inhibition of 86Rb+ efflux, were abolished in the absence of Ca2+. In the presence of 10 mM glucose, UL-DF 9 increased 86Rb+ and 45Ca2+ efflux from the islets, augmented the electrical activity in B-cells, and potentiated insulin release. These changes were suppressed by omission of extracellular Ca2+. Qualitatively similar effects were produced by lower concentrations of gliquidone itself. The data suggest that UL-DF 9 and gliquidone decrease the K+ permeability of the B-cell membrane, thereby causing a depolarization which leads to activation of voltage-dependent Ca channels and Ca2+ influx, and thus eventually increase insulin release. Hypoglycaemic sulfonylureas of the second generation therefore seem to contain a second chemical group that interacts with K channels of B-cells as does the sulfonylurea group itself.

Secretin and its C-terminal hexapeptide potentiates insulin release in mouse islets.

Peptides representing the C-terminal end of secretin were synthetized and their effects tested along with secretin on column-perifused isolated mouse pancreatic islets. Insulin release induced by 10 mmol/l D-glucose was potentiated by secretin tested in a concentration range of 0.01-10 micrograms/ml; the maximal effect was obtained with 1 microgram/ml secretin. This effect was mimicked by 50-500 micrograms/ml NH2-Leu-Leu-Gln-Gly-Leu-Val-NH2, [S-(22-27)], which represents an amidated C-terminal sequence of the secretin molecule. The consecutive smaller secretin C-terminal peptides had either no effects [Val-NH2, S-(24-27)] or only marginally [S-(26-27), S-(23-27)] potentiating effects on insulin release in the presence of 10 mmol/l D-glucose. The effects of secretin and S-(22-27) were not influenced by 2 mmol/l glutamine. The intact hormone and the five synthetic peptides as well as Val-NH2 had no stimulatory effect on islet glutamate dehydrogenase activity. In fact, S-(23-27), S-(24-27), and S-(25-27) inhibited the islet glutamate dehydrogenase activity, the activation by which amino acids and amino acid derivatives are known to elicit a potentiation of insulin release. Our results suggest that the C-terminal part is important to the marked potentiation of glucose-induced insulin release in vitro by secretin.

Effects of V.M.H. lesions on plasma insulin in the goose

The effects of bilateral electrolytic lesions of the ventromedial hypothalamus (V.M.H.) were examined in male geese. In addition to the known effects (hyperphagia, obesity and liver steatosis), V.M.H. lesions slightly increased plasma insulin level in the fasting and the fed state and largely enhanced insulin levels observed during an oral glucose and amino acid load. Therefore, V.M.H. lesions potentiate insulin release which may in turn participate in the development of hyperphagia and fattening in the goose as in mammals.

Mechanism of the stimulation of insulin release in vitro by HB 699, a benzoic acid derivative similar to the non-sulphonylurea moiety of glibenclamide.

HB 699 is a benzoic acid derivative similar to the non-sulphonylurea moiety of glibenclamide. The mechanisms whereby it affects B-cell function have been studied in vitro with mouse islets. In the presence of 3 mmol/l glucose, HB 699 decreased 86Rb+ efflux and accelerated 45Ca2+ efflux from islet cells, depolarized the B-cell membrane and induced an electrical activity similar to that triggered by stimulatory concentrations of glucose, and increased insulin release. The changes in 45Ca2+ efflux and insulin release, but not the inhibition of 86Rb+ efflux, were abolished in the absence of Ca2+. In the presence of 10 mmol/l glucose, HB 699 increased 86Rb+ and 45Ca2+ efflux from the islets, caused a persistent depolarization of the B-cell membrane with continuous electrical activity and markedly potentiated insulin release. All these changes were suppressed by omission of extracellular Ca2+. In the presence of 15 mmol/l glucose, diazoxide increased 86Rb+ efflux, hyperpolarized the B-cell membrane, suppressed electrical activity and inhibited insulin release. HB 699 reversed these effects of diazoxide. It is suggested that HB 699 decreases K+ permeability of the B-cell membrane, thereby causing a depolarization which leads to activation of voltage-dependent Ca channels and Ca2+ influx, and eventually increases insulin release. A sulphonylurea group is thus not a prerequisite to trigger the sequence of events that is also thought to underlie the releasing effects of tolbutamide and glibenclamide.

Sparteine increases insulin release by decreasing the K + permeability of the B-cell membrane

The effects of sparteine on the pancreatic B-cell function have been studied with mouse islets. In the presence of a non-stimulatory concentration of glucose (3 mM), sparteine (0.2–1 mM) decreased the rate of 86Rb + efflux from islet cells, depolarized the B-cell membrane, induced a glucose-like electrical activity and stimulated insulin release. This increase in release was observed over a large range of glucose concentrations (3–20 mM), and was most marked in the presence of 10 mM glucose. At this concentration of glucose, the effect of sparteine was already detected with 0.02 mM and was maximal with 0.5 mM. Higher concentrations of sparteine only had a transient effect on insulin release. In the presence of 10 mM glucose, 0.2 mM sparteine decreased 86Rb + efflux and increased 45Ca 2+ efflux from islet cells. The effect on 86Rb + efflux was only transient in the presence of extracellular calcium, whereas the effect on 45Ca 2+ efflux required the presence of extracellular calcium. The electrical activity induced by glucose in B-cells was augmented by sparteine which, at a concentration of 0.5 mM, produced a persistent depolarization with continuous spike activity. The potentiation of insulin release by sparteine was not reversible, but was inhibited by adrenaline and completely blocked by omission of extracellular calcium. Sparteine reversed the increase in 86Rb + efflux and the decrease in insulin release caused by diazoxide. These results show that sparteine increases insulin release by reducing the K + -permeability of the B-cell membrane.

Substrates for cyclic AMP-dependent protein kinase in islets of Langerhans. Studies with forskolin and catalytic subunit.

To investigate substrates for cyclic AMP-dependent protein kinase in intact islets of Langerhans, batches of islets were incubated with [32P]Pi for 1 h in the presence of 10 mM-glucose; the adenylate cyclase activator forskolin, which in parallel experiments was shown to increase islet cyclic AMP content and insulin release, was then added. Islets were homogenized and subcellular fractions prepared by differential centrifugation. Phosphopeptides were electrophoresed on sodium dodecyl sulphate/polyacrylamide gels and quantified by autoradiography and densitometry. Within 5 min forskolin caused increased labelling of Mr-25 000 and -30 000 cytosolic and Mr-23 000 and -32 000 particulate peptides; a rapid decrease in phosphorylation of Mr-18 000 and -34 000 cytosolic peptides was also observed. In addition, rather slower phosphorylation occurred of the Mr-15 000 peptide previously identified as histone H3 [Christie & Ashcroft (1984) Biochem. J. 218, 87-99]. When similar subcellular fractions were incubated with [gamma-32P]ATP and purified catalytic subunit of cyclic AMP-dependent protein kinase, peptides phosphorylated included cytosolic species of Mr 25 000 and 30 000 and particulate species of Mr 23 000 and 32 000. The distribution of RNA in the subcellular fractions suggested that the Mr-32 000 species could be a ribosomal protein. The 24 000 g pellet was heterogeneous, as judged by marker assays, and was therefore fractionated further by Percoll-density-gradient centrifugation. The peak containing the Mr-23 000 peptide was resolved from marker enzymes for plasma membranes, mitochondria and endoplasmic reticulum and coincided with a peak for insulin: hence the Mr-23 000 peptide is likely to be a secretory-granule component. The study demonstrates that the potentiation of insulin release that occurs when islet cyclic AMP is increased is accompanied by rapid phosphorylation of specific islet substrates for cyclic AMP-dependent protein kinase. The data are consistent with the hypothesis that protein phosphorylation is involved in the regulation of insulin secretion.

Stimulation of protein kinase C and insulin release by 1-oleoyl-2-acetyl-glycerol.

The membrane-accessible diacylglycerol 1-oleoyl-2-acetyl-sn-glycerol (OAG, 5-500 microM) caused a dose-related activation of protein kinase C in rat islet homogenates. In islet cell membranes exposed to [gamma-32P]ATP, OAG (100 microM) stimulated the net production of labelled phosphatidate and inhibited that of labelled phosphatidylinositol 4-phosphate. In intact islets exposed to 5.6 mM D-glucose, OAG (100 microM) decreased the outflow of 86Rb, increased that of 45Ca and caused a rapid stimulation of insulin release. The secretory response to OAG was dose-related in the 50-500 microM range, being most marked, in relative terms, at a glucose concentration close to the threshold value for stimulation of insulin release by this hexose. It was decreased but not abolished in the absence of CaCl2 and presence of EGTA. At variance with tumor-promoting phorbol esters, OAG failed to potentiate insulin release stimulated by a hypoglycaemic sulphonylurea. Although these findings support the view that activation of protein kinase C by diacylglycerol represents an efficient modality for stimulation of insulin release, they suggest that the effect of OAG upon islet function may not be solely attributable to such an activation.

Secretin potentiated insulin release. Structure-activity study with synthetic peptides

Secretin potentiated insulin release. Structure-activity study with synthetic peptides

Mechanisms of synergism between glucose and cAMP on stimulation of insulin release.

The mechanism of synergism between glucose and adenosine 3',5'-cyclic monophosphate (cAMP) on insulin release has been studied. Synergism may result from 1) inhibition of Na+-Ca2+ exchange by glucose and 2) a cAMP-induced sensitization of the release machinery to Ca2+. To distinguish between these two possibilities, isolated rat pancreatic islets were perifused with agents that raise intracellular levels of cAMP [3-isobutyl-1-methylxanthine (IBMX) and forskolin] and others that increase intracellular concentrations of Ca2+ either by blocking Na2+-Ca2+ exchange (ouabain and choline-Ringer solution) or by causing increased Ca2+ influx (KCl, carbachol, and 10 mM Ca2+). The results indicate that both the combination of cAMP and increased Ca2+ influx or blocked Na2-Ca2+ exchange and increased Ca2+ influx potentiated insulin release. When the relative potentiating abilities of cAMP and blocked Na2+-Ca2+ exchange were compared by determining the individual effects of IBMX and 1 mM ouabain (a concentration that causes similar inhibition of 45C2+ efflux as 16.7 mM glucose) in the presence of carbachol, cAMP was only 1.4 times more potent as a potentiating agent than blocked Na+-Ca2+ exchange. The greatest potentiation of insulin release was observed when Na+-Ca2+ exchange was blocked in the presence of increased levels of intracellular cAMP.

The ionic, electrical, and secretory effects of endogenous cyclic adenosine monophosphate in mouse pancreatic B cells: studies with forskolin.

Forskolin, an activator of adenylate cyclase, was used to study the effects of endogenous cAMP on the stimulus-secretion coupling in mouse pancreatic B cells. Forskolin produced a rapid, dose-dependent (0.05-50 microM) increase in islet cAMP, which was not influenced by the prevailing concentration of glucose and did not require extracellular Ca2+. At a nonstimulatory concentration of glucose (3 mM), a high concentration of forskolin (20 microM) barely doubled basal insulin release, marginally decreased 86Rb+ efflux from the islets, was without effect on the B cell membrane potential, and did not affect 45Ca2+ uptake (5 min). High concentrations of endogenously formed cAMP thus failed to mimic these early steps of the B cell response to glucose and other stimuli. At a threshold concentration of the sugar (7 mM), 5 microM forskolin slightly depolarized the B cell membrane, induced electrical activity (slow waves and spikes), stimulated 45Ca2+ uptake, and triggered insulin release. At a stimulatory concentration of glucose (10 mM), forskolin potentiated insulin release; half maximal and maximal effects were observed at 1 and 20 microM, respectively. Forskolin also increased the rate of 45Ca2+ and 86Rb+ efflux from islet cells, augmented 45Ca2+ uptake, and potentiated the electrical activity triggered by glucose in B cells. Its effects on insulin release, 45Ca2+ fluxes, and electrical activity were inhibited by omission of extracellular Ca2+ and/or Ca channels blockers. At a high concentration of glucose (25 mM), forskolin augmented the amplitude and the duration of the spikes occurring in the depolarized B cell membrane, slightly increased 45Ca2+ uptake, and potentiated insulin release. At threshold or stimulatory concentrations of glucose, endogenously formed cAMP can thus induce or enhance the Ca2+-dependent events normally triggered by the sugar. It is suggested that, besides its action on intracellular Ca stores, cAMP facilitates Ca2+ influx in B cells, by modulating the gating properties of the Ca channels.

Effects of theophylline and dibutyryl cyclic adenosine monophosphate on the membrane potential of mouse pancreatic beta‐cells.

The effects of theophylline and dibutyryl cyclic AMP on the membrane potential of mouse beta-cells were studied with micro-electrodes. They were compared to their effects on insulin release by perifused mouse islets. In 3 mM-glucose, theophylline (10 mM) depolarized the beta-cell membrane and stimulated insulin release, but did not induce electrical activity. Dibutyryl cyclic AMP (1 mM) was without effect. In 7 mM-glucose, theophylline (0.5-2 mM) and dibutyryl cyclic AMP (1 mM) slightly depolarized the beta-cell membrane, induced electrical activity in otherwise silent cells and increased insulin release. A higher concentration of theophylline (10 mM) hyperpolarized the beta-cell membrane, did not induce electrical activity, but also stimulated insulin release. In 10 mM-glucose, the membrane potential of beta-cells exhibited repetitive slow waves with bursts of spikes on the plateau. Under steady state, these slow waves were differently affected by low or high concentrations of theophylline. At 0.5-2 mM, theophylline shortened the intervals, lengthened the slow waves and slightly increased their frequency. On the other hand, 10 mM-theophylline markedly decreased the duration of both intervals and slow waves, and increased their frequency. The effects of 1 mM-dibutyryl cyclic AMP were similar to those of 2 mM-theophylline. With 2-10 mM-theophylline, two other effects were also observed: a transient hyperpolarization with suppression of electrical activity immediately after addition of the methylxanthine and an increase in electrical activity upon its withdrawal. Theophylline and dibutyryl cyclic AMP markedly potentiated insulin release induced by 10 mM-glucose. The magnitude of these changes did not correlate well with the importance of the changes in electrical activity. However, with 2-10 mM-theophylline the increase in release was also preceded by an initial transient inhibition, whereas withdrawal of the methylxanthine was accompanied by a further increase. When Ca influx was inhibited by D600, the slow waves were suppressed, the membrane was depolarized to the plateau level and only few spikes were present. Although theophylline markedly increased insulin release under these conditions, it did not affect the membrane potential. Several conclusions can be drawn from this study. Insulin release and electrical activity in beta-cells can be dissociated when intracellular Ca is used to trigger exocytosis. High concentrations of theophylline produce effects unrelated to cyclic AMP.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of lipoxygenase and cyclooxygenase inhibitors on glucose-stimulated insulin secretion from the isolated perfused rat pancreas

Some of the metabolites of arachidonic acid formed in the lipoxygenase and cyclooxygenase pathways stimulate insulin release. We studied the relative importance of each of these pathways in the modulation of glucose-induced insulin release by using inhibitors of arachidonate metabolism. Perfusion of the isolated rat pancreas with two chemically different inhibitors of cyclooxygenase, flurbiprofen and sodium salicylate, markedly inhibited prostaglandin E2 release, but had little effect on glucose-induced insulin release or on potentiation of insulin release caused by prior exposure to glucose. On the other hand, nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, not only inhibited both phases of glucose-induced insulin release but also abolished the potentiation effect. These effects of NDGA prevailed, when it was administered together with flurbiprofen, which caused profound inhibition of prostaglandin E2 release. We conclude that 1) lipoxygenase pathways play a dominant role in glucose-stimulated insulin release, and 2) endogenous lipoxygenase metabolites influence the potentiating effect of glucose on the release of insulin in response to a subsequent stimulation.

Unexpected potentiation of insulin release by the calcium store blocker TMB-8.

Insulin release from rat pancreatic islets, stimulated by cAMP, 3-isobutyl-1-methylxanthine, or forskolin, is potentiated by TMB-8 [8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate], a drug that blocks the efflux of calcium from intracellular calcium stores without affecting influx. There is a short latent period before the potentiation occurs which can be reduced by preincubation with the drug. TMB-8 alone neither stimulates nor inhibits insulin release. The results suggest that an intracellular store fills with calcium because of the blocked efflux and unchanged influx, and loses its ability to regulate the cytosol Ca++ concentration. Under basal conditions, in the presence or absence of TMB-8, the plasma membrane is able to regulate the cytosol Ca++ concentration at low levels so that no change in insulin release occurs. However, when 3-isobutyl-1-methylxanthine, cAMP, or forskolin add an additional Ca++ load to the cytosol, the regulator capacity of the plasma membrane is overwhelmed, and cytosol Ca++ rises to higher levels than in the absence of TMB-8 because the filled store is no longer regulating. Thus, insulin release is potentiated.

Effects of alloxan on the islets of Langerhans inhibition of leucine metabolism and insulin secretion

The action of alloxan on the metabolism of the islets of Langerhans was studied in vitro. Isolated mouse islets were exposed to the drug at 4°C to prevent its decomposition. Islet uptake of leucine was subsequently estimated at 37°C, and was found not to be affected by the drug. However, islet leucine oxidation was strongly inhibited by the preceding alloxan exposure. The islets were protected against this inhibition by an incubation at a high glucose concentration prior to alloxan exposure. In contrast, a high concentration of leucine failed to provide full protection of either islet leucine oxidation or islet glucose oxidation. Furthermore, it was shown that alloxan impeded islet insulin response to both leucine and glucose. In addition, the potentiation of insulin release by theophylline was abolished after alloxan treatment of the islets. The results reinforce the hypothesis that the B-cytotoxicity of alloxan reflects an interaction with intracellular sites involved in the oxidative metabolism of the B-cell, and that these sites may be protected against the action of the drug by some metabolite of glucose.

Pancreatic endocrine responses to exogenous neurotensin in the conscious calf.

1. Responses to neurotensin have been investigated in conscious calves 2-5 weeks after birth given continuous I.V. infusions of the peptide for 15 min (5 pmol. kg-1 . min-1). 2. In control calves the concentration of the peptide in the arterial plasma had risen by 160 +/- 10 pmol/l at the end of the infusion,. after which it fell exponentially (t1/2: 1.4 min). 3. This dose of neurotensin produced no significant change in mean heart rate, aortic blood pressure, plasma gastrin or glucose concentration. 4. It was found that neurotensin could produce a pronounced rise in the concentration of both insulin and pancreatic polypeptide (PP) in the arterial plasma, together with a much smaller rise in pancreatic glucagon concentration. 5. Each of these three pancreatic endocrine responses was found to be glucose-sensitive within the range ca. 5.0-9.0 mmol/l. Hyperglycaemia potentiated insulin release and inhibited release of PP and glucagon. 6. The results are discussed in relation to the findings of other workers in other species.

Actions of GIP

Two structurally similar peptides were isolated from a preparation of GIP using an HPLC system. The major peptide corresponds to GIP 1–42 and the minor has the sequence GIP 3–42. GIP 1–42 has both insulinotropic and somatostatinotropic activities, whereas GIP 3–42 has only insignificant activity. GIP was also shown to potentiate insulin release initiated by D-glyceraldehyde, L-leucine/L-glutamine and 2-keto-isocaproic acid. No potentiation was observed with 2-ketocaproate. The 4 substrates studied are all metabolized but via different mechanisms.

Insulin release, insulin sensitivity, and glucose intolerance.

Groups of subjects with different degrees of glucose intolerance were examined in order to determine, first, the capacity of the beta cells to release insulin upon glucose stimulation and, second, sensitivity to insulin. The groups were selected on the basis of fasting blood glucose value and tolerances to oral and intravenous glucose administration. The body weights, ages, and sexes of the subjects were well matched with those of control subjects with normal tolerances to oral and intravenous glucose administration. Computer analysis of the glucose and insulin curves during a standardized glucose infusion test made possible the measurement of the initiatory (parameters KI and IP) and potentiatory (parameter KP) effects of glucose on insulin release and of the sensitivity to endogenous insulin (parameter KG). In subjects with impaired oral but normal intravenous glucose tolerance tests, KG was decreased, KP was increased, and KI and IP were normal. However, in these subjects, KI and IP were considerably lower than in a matched group of control subjects with the same decrease in KG but with normal oral and intravenous glucose tolerance tets. In subjects in which both oral and intravenous glucose tolerance tests were impaired and in subjects with mild manifest diabetes, KI, IP, and KG were decreased whereas KP was normal. These data suggest that all stages of glucose intolerance are accompanied by a decreased ability of glucose to initiate insulin release and by decreased sensitivity to insulin. These derangements seem to be partially compensated for by enhancement of the capacity of glucose to potentiate insulin release in subjects with decreased oral but normal intravenous glucose tolerance tests.